CERTIFICATE

This is to certify that this project on

EXPERIMENTAL EMBRYOLOGY embodies all the effects and work done by ISMAT PERWEEN, EXAM roll no. 11MCRBS51003 a student of class MARWARI

B.SC. BOTANY HONS, SEM-III of

COLLEGE, Ranchi in practical fulfillment for the purpose of practical examination

PROJECT GUIDE

ACKNOWLEDGEMENT
It is a matter of great pleasure for me to acknowledge my feeling of extreme gratitude and sincere regards to our teachers for their regular encouragement and proper guidance. It was because of their steady guidance and inspiration that I could complete this work in its present form. I am obliged to professors for his continuous support and encouragement. This acknowledgement will remain incomplete if do not specially thanks to our particular teachers for their valuable suggestions and timely intervention. Specially thanks to my friends who kept me helping throughout the session and helped me all the way in completing my project work. By, NAME: - ISMAT PERWEEN ROLL NO. : - 11MCRBS51003 CLASS: -BSC , PART-II, SEM-III SESSION: -2011-2014

CONTENT
INTRODUCTION INDUCTION EXPERIMENTAL APPROACHES COMMITMENT COMPETENCE GROWTH FACTOR EMBRYO CULTURE CONCLUSION

INTRODUCTION Experimental embryology, a branch of embryology, is an important partner of descriptive embryology. Hypotheses about development are tested using genetic and embryo manipulations, e.g., lesions, transplantations, and transgenic organisms. Important concepts developed by experimental embryologists include induction, commitment, and programmed cell death. Experimental embryology addresses, for example, questions about mechanisms underlying observed developmental changes, including whether these changes are autonomous or conditional. Cell-autonomous (or mosaic) Development Observation of development in undisturbed embryos gives the impression that cells follow a genetic program to their cell fate, perhaps influenced by the portion of cytoplasm it inherited during cleavage. A cell's fate is thus largely determined by its lineage (Sidney Brenner's "European plan").

Ablation of one or more blastomeres may result in missing parts. Transplanted cells may develop as if they had not been moved. This type of development is thought to be important in most invertebrates.

Conditional (or regulative) Development Manipulation of embryos often demonstrates that cell fate is influenced by the cell's environment. Cellular behavior may be influenced by intercellular signaling molecules (e.g., morphogens), direct cell-cell interactions (e.g., via cell-adhesion molecules) and extracellular matrix. A cell's fate is thus largely determined by its neighbors (Brenner's "American plan").

Ablation of one or more blastomeres may have little effect on the final form of the organism.

Transplanted cells may develop differently, or influence the development of its new neighbors.

This type of development is thought to be important in vertebrates and some invertebrates (including most insects).

Induction Induction is the influence of an inductor (an "organizer" or "evocator") on the differentiation of adjacent cells or the development of an embryonic structure. Differentiation under the influence of an organizer or other "positional information" is an important component of theories of pattern formation in

morphogenesis. An organizer is a group of cells that influence the growth and development of adjacent (competent) cells, through the effects of an evocator.

Competence Competence is the ability of a cell to respond in a specific manner to an inductor. For example, competence may depend on the expression of a specific cell-surface receptor for an evocator. Experimental Approaches Hans Spemann received a Nobel Prize in 1935 for discovery of the organizer effect in embryonic development.

Organizers are traditionally characterized by the effects of their ablation on surrounding tissues, or the effects of their transplantation on surrounding host tissues.

Tests of potential evocators include injection into embryos and studies of transgenic mice.

Commitment Differentiation is an overt change in cellular biochemistry, structure and function. It is preceded by the covert commitment of cells to particular fates. Commitment is divided into two stages, specification and determination. Specification are early, reversible steps; whereas determination implies irreversibility.

Potential Potency is the extent of the potential of a developing cell to differentiate into a variety of cell types. During development, the potency of cells is generally reduced as they become differentiated. Potency and commitment have an inverse relationship. The prospective potency of an embryonic cell (the set of cell types that could arise from it) is greater than its prospective fate (the set of cell types that would be produced in normal, unaltered development).

Embryonic stem cells are totipotential cells (able to differentiate into any type of cell).

Most stem cells are pluripotential cells (able to differentiate into many cell types).

Progenitor cells include multipotential cells (able to differentiate into several cell types) and committed cells.

Experimental Approaches The standard experiments to test cellular commitment and potential include transplantation and ablation.

Tissues can be destroyed to determine if there are any changes in the differentiation of tissues surrounding the lesion.

Tissues can be moved to a different location (in the same or a different individual) to test their commitment and potential, and to examine the effects of graft and host tissues on each other.

Programmed Cell Death (Apoptosis) Perhaps surprisingly, cell death is a normal developmental (morphogenetic) process. If cells are no longer needed, they commit suicide by activating an intracellular death program, called programmed cell death or apoptosis. Well studied examples of apotosis in normal vertebrate development include:

Sculpting the digits in the developing mouse paw by apoptosis. Interdigital tissue between digital rays in the paw plate is removed by apoptosis, probably induced by bone morphogenetic proteins.

In the frog spinal cord, more neurons are produced than are needed. This strategy of overproduction followed by culling ensures that all target cells are contacted by nerve cells and that extra nerve cells are eliminated.

Growth factors Cell survival may be dependent upon the presence of growth factors. For example, survival of spinal cord motor neurons depends on nerve growth factor released by their target cells (muscle cells). Growth factors are also important inducers of proliferation and differentiation. Experimental approaches

John Sulston shared a Nobel Prize in 2002 for his description of cell lineage in development of the nematode worm C. elegans. He described the pattern of cell proliferation that resulted in adults containing exactly 959 cells, and found that during development another 131 cells died.

H. Robert Horvitz shared a Nobel Prize in 2002 for his work on genetic analysis of programmed cell death in development of C. elegans. He discovered and characterized genes involved in programmed cell death in C. elegans, and showed that similar genes are present in humans.

Stanley Cohen and Rita Levi-Montalcini shared a Nobel Prize in 1986 for the discovery and and isolation of growth factors (Nerve growth factor and Epidermal growth factor).

Embryo Culture In addition to root, shoot, and pollen culture, embryo culture has also been done for the production of haploid plants. Embryo culture is used for the recovery of plants from distinct crosses. Embryo culture is useful where embryo fails to develop due to degeneration of embryonic tissues. It is being used extensively in the extraction of haploid barley (Hordeum vulgare) from the crosses H. vulgare x H. bulbosum. Embryo culture is also a routine technique employed in orchid propagation and in breeding of those species that show dormancy. Das and Barman (1992) developed the method of regeneration of tea shoots from embryo callus. The embryo callus produced somatic embryoids within 8 weeks of culture in the second medium which differentiated into buds after 2 weeks. Several shoots with 4-6 leaves developed after 16 weeks of culture.

Culturing method The general method of embryo culture follows the following steps.

(i)

Pluck healthy and mature fruits from the field and wash thoroughly in running water for about an hour.

(ii)

Surface sterilize with 0.01% Tween-20 for 15 min, rinse seeds several times with distilled water and finally treat with 0.01% HgCl2 solution for 10-15 min. Finally rinse it for six times with sterile distilled water.

(iii)

Break seeds aseptically and isolate the embryo.

(iv)

Culture embryo on callus proliferation medium. Supplement the basal medium of Murashige and Skoog (1962) with different combinations and concentrations of sugar, vitamins, hormones and other growth adjuvants for callus proliferation and shoot regeneration.

(v)

Incubate the cultures at 22-25C under a 16 h

photoperiod of 2000 lux luminous intensity. (vi) After two weeks of inoculation the embryo begins to swell on callus proliferation medium. Distinct callus growth is observed after 4 weeks. (vii) After 8 weeks of inoculation transfer the callus on shoot regeneration medium. Within 4 weeks of transfer into second medium the callus turns green and produces soft spongy tissue. Some of these tissues are differentiated into embryoids. (viii) The embryoids produce cluster of budlets when subcultured onto shoot regeneration medium. The budlets grow into shoots and produce 2-3 leaf appendages within 12 weeks. Thereafter, they are separated into individual shoots and then subcultured into a fresh medium of the same composition until

shoots develop.

CONCLUSION a branch of embryology that studies the mechanisms controlling the individual development of animals and plants by means of experiments on living organisms. It uses such methods as marking, removal, transplantation, and isolation of body parts and organs. It also studies the action of various external factors on embryonic development. Experimental embryology identifies the stages of the determination of the material of rudimentary organs and tissues, the sources of formative or inductive influences, the role of synthesis of macromolecules in the processes of determination and differentiation, and the factors responsible for morphogenesis. By removing, inactivating, or transplanting cell nuclei, experimental embryologists investigate the interaction of the nucleus and cytoplasm during gametogenesis and embryonic development, as well as the stages and factors of differential activation of genes in the course of development.